Name: Dendra2 Photoswitching through the Mammary Imaging Window (Video) | JoVE Journal
Uploaded: 2009-06-05T00:00:00.000+00:00
Duration: 9 min 12 s
Description: Learn intravital photoswitching of Dendra2-labeled tumor cells in mouse mammary fat pad using a specialized imaging window. This protocol enables tracking metastatic behavior over several days.

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This work was supported by US Department of Defense (BC075554 to B.G. and BC061403 to D.K.), US National Institutes of Health (U54GM064346 to J.v.R.; CA100324 to J.C., J.E.S. and J.W.; CA77522 to J.E.S., U54CA126511 to J.C. and B.G.). We thank D. Entenberg for help with microscopy, M. Rottenkolber for help in fabrication of the imaging box and J. Pollard (Albert Einstein College of Medicine) for the F4/80 antibody.

1. Generation of fluorescent tumors using injection of tumor cells into the mammary fat pad:
<ol>
<li>Grow a cell line expressing Dendra2 protein (as a cytoplasmic marker) to 40 - 80% confluency.</li>
<li>Rinse dishes at least 3 times with PBS w/o Ca2+ or Mg2+.</li>
<li>Add 3 ml of trypsin per 10 cm dish and incubate at 37 &deg;C until most of the cells detach and then hit the dish against a flat surface to shake it.</li>
<li>Rinse all the cells off the dish, and use a scraper (rubber policeman) to collect matrix as well.&nbsp; Add the 5 ml of PBS.&nbsp;Take an aliquot to count during centrifugation.</li>
<li>Centrifuge at 800 g for 5 min.</li>
<li>Aspirate and resuspend in PBS to a concentration of 5- 10x 106/ml.&nbsp; Store on ice until injected (inject within 30 min).</li>
<li>Place a cage containing 4-5 week old female immune deficient mice (e.g. SCID) inside a sterile hood.</li>
<li>Spray the area around the 4th (abdominal) nipple with 70% ethanol.</li>
<li>Inject 0.1 ml into mammary fat pad. If an assistant can be found, one person can hold the mouse in place while the other inserts the needle inside the mammary fat pad. If you are injecting by yourself, put the mouse under light anesthesia using isoflurane.</li>
<li>Once the tumor has grown to 5-7 mm, the Mammary Imaging Window (MIW) should be inserted. Note: Alternatively, for some applications, the MIW can be inserted on top of the healthy mammary fat pad and the cells can be injected afterwards. That approach allows imaging of transiently transfected cells in a physiological environment.</li>
</ol>
2. Manual fabrication of the Mammary Imaging Window (MIW):
<ol>
<li>MIWs (Figure 1A) are made of tissue grade plastic to guarantee biocompatibility. For manual construction, we use 5 or 10 cm dishes.</li>
<li>Put latex gloves on.</li>
<li>Heat up a tissue-culture dish and creat a curved surface by pushing a rounded, hard object against the heated dish (we use a 1-inch dremmel bit for this).</li>
<li>Cut out the center of the dish using a heated razor blade. </li>
<li>Use a small, cone-shaped dremmel bit to make a hole in the center of the dome-shaped plastic base (6-7mm in diameter). </li>
<li>Make the edges of the plastic base completely smooth by sanding it with the dremmel and further filing it. </li>
<li>File the top of the plastic base making a flat surface for the glass coverslip. The diameter of the filed, flat surface should be 9-10mm.</li>
<li>Glue the 8mm circular glass coverslip (number 1) on the flattened surface using the superglue (cyanoacrylate adhesive).</li>
<li>Wait for the glue to dry (15 minutes).</li>
<li>Use a heated 26G needle to make eight suturing holes puncturing from the outside (where the glass is) to the inside of the base. Holes should be evenly distributed around the coverslip, .5-1mm away from the edge of the coverslip. </li>
<li>Widen the holes using a 5-0 suturing needle.</li>
<li>Puncturing holes in the plastic base will make the inner surface uneven. Using sand paper, make this surface completely smooth.</li>
<li>Change the gloves and brush of small plastic particles from the MIW using a small brush.</li>
<li>Wash the MIW with deionized water.</li>
<li>Wash the MIW using 70% ethanol. Use a Q-tip to clean the glass so it is completely transparent. If there are foggy spots present from the glue vapor, carefully use acetone with a Q-tip.</li>
<li>Sterilize the MIW by UV exposure for 3 h at each side.</li>
</ol>
Semi-Manual fabrication of the Mammary Imaging Window (MIW):
In order to fabricate the plastic base, we are currently using silicone rubber casting molds created using hand-made MIWs. The mold is comprised of two parts of silicone rubber that make an exact replica of both the front and back of the original when filled with polyester resin and then immediately joined together.&nbsp;The liquid polyester resin is mixed together 9:10 and results in a hard structure when fully cured in 48h. The plastic is UV protected and archival without breaking down or becoming yellow over time. After the base is cured, steps 2.8-2.16 are done the same way as for manually-made MIWs.
3. Insertion of the MIW:
<ol>
<li>The area around the 4th nipple should have a small (5-7 mm diameter) tumor several days/weeks after injection of tumor cells depending on the cell type (Figure 1B). How much time has passed after injection of cells expressing Dendra2 photoswitchable protein or other fluorescent proteins depends on the cell line used. The tumor should not be visibly necrotic, and should have an intact skin with hair on top of the tumor. Mice with necrotic tumors should be euthanized.</li>
<li>Prepare the sterile space for the surgery- lay down a piece of sterile cloth inside a sterile hood. Put a piece of sterile gauze in the middle, and lay a few MIWs and sterile instruments around it: we use small standard scissors, spring scissors, microdissecting tweezers, microdissecting forceps, needle holder. Keep sterile Q-tips, a bottle of 70% ethanol and sterile gloves in the corner of the hood.</li>
<li>The mouse is anesthetized in the sterile hood using IP injection of 2.5% avertin (20 &mu;l/g) in HBSS or, alternatively 10 &mu;g/kg ketamine + 10 &mu;g/g xylazine (contact your Animal Care Institute for approvals and ordering). Note: Avertin solution should be prepared bi-weekly, filter sterilized using a 0.22 &mu;m filter and stored as 500 &mu;l aliquots at 4 &deg;C (in the dark).</li>
<li>Remove hair by shaving the area above the tumor using a small animal shaver.</li>
<li>Remove the rest of the hair using Nair hair removal cream (available in any drugstore). Clean the skin using ethanol-dipped Q-tips.</li>
<li>Transfer the animal onto the sterile gauze and apply ophthalmic ointment to the eyes to keep them from drying and infection.</li>
<li>Sterilize the skin with betadine and clean with 70% ethanol.</li>
<li>Transfer the animal onto a sterile surgical cloth inside the sterile hood. </li>
<li>Pull the skin immediately medial to the nipple using forceps and cut ~2mm incision in the skin.</li>
<li>Separate the underlying mammary fat pad from the skin using dissection scissors and forceps. The skin and hole stretch during this separation step to a size that will accommodate insertion of the MIW.</li>
<li>If accidently a vessel is hit during sugery, use a sterile Q-tips to remove any blood that is produced/to stop the bleeding from becoming excessive. </li>
<li>Insert the MIW such that there is skin on top of the MIW base and suture in place using non-absorbable thread and reverse cutting needle.</li>
<li>Use tissue adhesive or cyanoacrylate to fill in the suturing holes and secure the MIW to the skin.</li>
<li>Add TMP-SMX antibiotic mix: (Sulfamethoxazole 0.6 mg/ml, Trimethoprim 0.12 mg/ml) into the cage water bottle for 3 days before and after the surgery. </li>
<li>The animal stays under anesthesia for 1-4h after avertin IP injection. Since the surgery commonly takes ~45min, it is important to help the animal recover afterwards:</li>
<li>Place a hand heating pad (37 &deg;C) (available in drugstores) on the bottom of the cage and cover it with gauze.</li>
<li>Keep the animal on top of the pad until it turns on its stomach and starts walking.</li>
<li>If the animal is unconscious for more than 3h, inject 0.3 ml of HBSS intraperitoneally for rehydration.</li>
<li>The animal is allowed to recover over the next 3-4 days before the first imaging session takes place.</li>
</ol>
4. Imaging Box Construction
The imaging box assures that the MIW is sitting flat, right above the microscope objective. It also facilitates temperature and anesthesia control through constant airflow of isoflurane. The box is made of plexiglass and glued together using plastic weld. It is fitted for the specific microscope stage and therefore the shape of its bottom varies depending on the microscope used (Figure 2 shows the scheme of the box fitted to Leica SP5 microscope stage). Dimensions of the box in centimeters are l=11.4 cm, w=7.6 cm, h=4.4 cm (Figure 2A). The bottom of the box consists of a hollow plate, 12.7x8.4 cm in size, which fits inside the Leica SP5 microscope stage, and two sliding &ldquo;doors&rdquo;, 5.7 X 5.7 cm_each, which form a circular opening (d=2.22 cm) when closed. The MIW fits into this opening. The front piece contains the inlet hole for the anesthesia delivery while one of the side pieces contains an outlet hole which leads to the vacuum. Note that the condenser and the slide holder need to be removed prior to the imaging box placement.
5. Imaging Box Use
<ol>
<li>Place the animal under anesthesia with isoflurane and apply ophthalmic ointment to the eyes.</li>
<li>Attach the isoflurane exhaust from the anesthesia machine to the imaging box front, on the outside of the inlet hole (Figure 2B).</li>
<li>Attach a second tube to the outlet hole of the imaging box. This tube should be attached to the gas filter and then to the vacuum. </li>
<li>Open the box lid and open the box doors and place the animal, MIW side facing the bottom, and sliding the doors into the box.</li>
<li>Hold the box and adjust the bottom doors so that the MIW base is held and immobilized between them. The MIW base should be at the same level as the imaging box doors, while the MIW coverslip should be just a few millimeters below this level. Make sure that the positioning of the MIW between the bottom sliding doors ensures that the coverslip (glass portion of the MIW) lies flat, parallel to the bottom doors the box. This will ensure proper focusing. </li>
<li>Lower the microscope objective, to allow space for placement of the box on top of the microscope stage.</li>
<li>Place the box on the microscope stage, and adjust the stage so that the MIW coverslip is above the objective. </li>
<li>Focus the objective and start imaging.</li>
<li>While using isoflurane, the animal should be monitored by visual inspection of the breathing frequency. This can be done visually, or by monitoring the frequency of breathing artifacts appearing during imaging collection (these can interfere with data acquisition). The MouseOx pulse oximetry system (Starr life Sciences Corp) has been used succesfully. That way the data can be gathered continuously without turning the lights on in the room to check on the animal. Note: After each imaging session, animal recovery should be facilitated by wrapping a small hand heating pad in gauze or kimwipes and putting it under the animal in the cage. Note: If an immersion objective is used to image through the MIW, the immersion medium (glycerol, water, oil) should be cleaned off the imaging window. The MIW should be inspected for any cracks or other damage that might have occurred during imaging. This is critical for collecting data during multiple imaging sessions. </li>
</ol>
6. Photoswitching and imaging of Dendra2-labeled cells
The procedure is described for the Leica SP5 confocal microscope; the laser power at the focal plane, available objectives and software options vary when using other confocal microscopes or multiphoton imaging set up, and therefore the protocol described below should be used as a guide to optimize the experiment on your microscope.
<ol>
<li>By observing the tumor through the eyepiece (10x) with the green fluorescence filter, locate major blood vessels which are visibly flowing and laying flat in the same focal plane.</li>
<li>Position one of the vessels in the center of the field and switch to PMT detection.</li>
<li>Set up sequential imaging collection for two channels. One of the channels uses 488nm laser line (10% power), and collects scattering from extracellular matrix (480-495nm) and emission from the green form of Dendra2 (505-540nm). The second channel uses the 543nm laser line (90% power) and collects emission from the red form of Dendra2 (555-600nm). Collect 3D images pre-photoswitching.</li>
<li>Use the ROI scan option to photoswitch a chosen population of cells using the 405nm laser line (30% power, 20-40 scans). Collect emission of the red form of the protein to monitor the switching.</li>
<li>Using the same sequential routine as in 6.3, collect post-switching 3D images.</li>
<li>If photoswitching more than one region in the tumor, it is advised to take pictures through the oculars using a digital camera, in both green and red channels (Figure 3A, B). This will help in the orientation during the subsequent imaging sessions. Note: Additionally, blood vessels can be temporarily labeled using tail-vein injection of fluorescent dextran (Cascade Blue or Alexa Fluor 647, both 10kDa). A few hours after the injection, dextrans will leave the blood vessels and permanently label the macrophages.</li>
</ol>
Representative Results:
Figure 3C shows a rectangular photoswitched region (red) oriented orthogonally relative to the blood vessel (no fluorescence). Non-photoswitched cells are green, while the scattering from the extracellular matrix is purple. Image is a maximum intensity projection of four images along the Z-axis (20-50 &mu;m depth).

<ol><li>Farina, K. L., Wyckoff, J. B., Rivera, J., Lee, H., Segall, J. E., Condeelis, J. S., Jones, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+motility+of+tumor+cells+visualized+in+living+intact+primary+tumors+using+green+fluorescent+protein%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">Cell motility of tumor cells visualized in living intact primary tumors using green fluorescent protein.</a> Cancer Res. 58, 2528-2532 (1998).</li>
<li>Naumov, G. N., Wilson, S. M., MacDonald, I., Schmidt, E. E., Morris, V. L., Groom, A. C., Hoffman, R. M., Chambers, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cellular+expression+of+green+fluorescent+protein%2C+coupled+with+high-resolution+in+vivo+videomicroscopy%2C+to+monitor+steps+in+tumor+metastasis%5BTitle%5D)+AND+%22J+Cell+Sci%22%5BJournal%5D)">Cellular expression of green fluorescent protein, coupled with high-resolution in vivo videomicroscopy, to monitor steps in tumor metastasis.</a> J Cell Sci. 112, 1835-1842 (1999).</li>
<li>Brown, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+measurement+of+gene+expression%2C+angiogenesis+and+physiological+function+in+tumors+using+multiphoton+laser+scanning+microscopy%5BTitle%5D)+AND+%22Nature+Med%22%5BJournal%5D)">In vivo measurement of gene expression, angiogenesis and physiological function in tumors using multiphoton laser scanning microscopy.</a> Nature Med. 7, 864-868 (2001).</li>
<li>Wang, W., Wyckoff, J. B., Frohlich, V. C., Oleynikov, Y., Huttelmaier, S., Zavadil, J., Cermak, L., Bottinger, E. P., Singer, R. H., White, J. G., Segall, J. E., Condeelis, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single+cell+behavior+in+metastatic+primary+mammary+tumors+correlated+with+gene+expression+patterns+revealed+by+molecular+profiling%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">Single cell behavior in metastatic primary mammary tumors correlated with gene expression patterns revealed by molecular profiling.</a> Cancer Res. 62, 6278-6288 (2002).</li>
<li>Sidani, M., Wyckoff, J., Xue, C., Segall, J. E., Condeelis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Probing+the+microenvironment+of+mammary+tumors+using+multiphoton+microscopy%5BTitle%5D)+AND+%22J+Mammary+Gland+Biol+Neoplasia%22%5BJournal%5D)">Probing the microenvironment of mammary tumors using multiphoton microscopy.</a> J Mammary Gland Biol Neoplasia. 11, 151-163 (2006).</li>
<li>Wyckoff, J. B., Wang, Y., Lin, E. Y., Li, J. F., Goswami, S., Stanley, E. R., Segall, J. E., Pollard, J. W., Condeelis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+visualization+of+macrophage-assisted+tumor+cell+intravasation+in+mammary+tumors%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">Direct visualization of macrophage-assisted tumor cell intravasation in mammary tumors.</a> Cancer Res. 67, 2649-2656 (2007).</li>
<li>Egeblad, M., Ewald, A. J., Askautrud, H. A., Truitt, M. L., Welm, B. E., Bainbridge, E., Peeters, G., Krummel, M. F., Werb, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Visualizing+stromal+cell+dynamics+in+different+tumor+microenvironments+by+spinning+disk+confocal+microscopy%5BTitle%5D)+AND+%22Dis+Model+Mech%22%5BJournal%5D)">Visualizing stromal cell dynamics in different tumor microenvironments by spinning disk confocal microscopy.</a> Dis Model Mech. 1, 155-167 (2008).</li>
<li>Mempel, T. R., Henrickson, S. E., von Adrian, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((T-cell+priming+by+dendritic+cells+in+lymph+nodes+occurs+in+three+distinct+phases%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">T-cell priming by dendritic cells in lymph nodes occurs in three distinct phases.</a> Nature. 427, 154-159 (2004).</li>
<li>Miller, M. J., Wei, S. H., Cahalan, M. D., Parker, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Autonomous+T+cell+trafficking+examined+in+vivo+with+intravital+two-photon+microscopy%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+USA%22%5BJournal%5D)">Autonomous T cell trafficking examined in vivo with intravital two-photon microscopy.</a> Proc Natl Acad Sci USA. 100, 2604-2609 (2003).</li>
<li>Kedrin, D., Gligorijevic, B., Wyckoff, J., Verkhusha, V. V., Condeelis, J., Segall, J. E., van Rheenen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intravital+imaging+of+metastatic+behavior+through+a+mammary+imaging+window%5BTitle%5D)+AND+%22Nat.+Methods%22%5BJournal%5D)">Intravital imaging of metastatic behavior through a mammary imaging window.</a> Nat. Methods. 5, 1019-1021 (2008).</li>
<li>Chudakov, D. M., Lukyanov, S., Lukyanov, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tracking+intracellular+protein+movements+using+photoswitchable+fluorescent+proteins+PS-CFP2+and+Dendra2%5BTitle%5D)+AND+%22Nat.+Protocols%22%5BJournal%5D)">Tracking intracellular protein movements using photoswitchable fluorescent proteins PS-CFP2 and Dendra2.</a> Nat. Protocols. 2, 2024-2032 (2007).</li>
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<table><tbody><tr><td>PBS w/o Ca, Mg</td><td>GIBCO, by Life Technologies</td><td>14190-144</td><td></td></tr><tr><td>Trypsin</td><td>GIBCO, by Life Technologies</td><td>25300</td><td></td></tr><tr><td>HBSS</td><td>GIBCO, by Life Technologies</td><td>14025</td><td></td></tr><tr><td>DMEM media (MDA-MB-231 cells)</td><td>GIBCO, by Life Technologies</td><td>11965</td><td></td></tr><tr><td>Isoflurane(Aerrane)</td><td>Baxter Internationl Inc.</td><td># NDC 10019-773-40</td><td></td></tr><tr><td>SCID mouse</td><td>National Cancer Institute</td><td>N/A</td><td></td></tr><tr><td>Culture dishes</td><td>BD Biosciences</td><td>353003</td><td>Custom Order</td></tr><tr><td>Circular coverslip 8 mm</td><td>Scientific, Inc.</td><td>8CIR-1-FIS</td><td></td></tr></tbody></table>

dendra2 photoswitching through the mammary imaging window

Dendra2 Photoswitching through the Mammary Imaging Window

In the last decade, intravital microscopy of breast tumors in mice and rats at single-cell resolution1-4 has resulted in important insights into mechanisms of metastatic behavior such as migration, invasion and intravasation of tumor cells5, 6, angiogenesis3 and immune cells response7-9. We have recently reported a technique to image orthotopic mammary carcinomas over multiple intravital imaging sessions in living mice10. For this, we have developed a Mammary Imaging Window (MIW) and optimized imaging parameters for Dendra211 photoswitching and imaging in vivo. Here, we describe the protocol for the manufacturing of MIW, insertion of the MIW on top of a tumor and imaging of the Dendra2- labeled tumor cells using a custom built imaging box. This protocol can be used to image the metastatic behavior of tumor cells in distinct microenvironments in tumors and allows for long term imaging of blood vessels, tumor cells and host cells.

Intravital photoswitching and tracking of Dendra2-labeled tumor cells through the Mammary Imaging Window is a technique which allows us to image the metastatic behavior of tumor cells in chosen tumor microenvironments over a timescale of days.

In the last decade, intravital microscopy of breast tumors in mice and rats at single-cell resolution1-4  has resulted in important insights into ...

This video illustrates a protocol beginning with the injection of a fluorescent protein expressing tumor cell line into the mouse mammary fat pad. A specialized chamber for in vivo imaging is constructed and then surgically implanted into the animal at which point imaging data can be acquired. Hi, I'm Dr.Vic from the laboratory of Dr.John Condi and Dr.Jeffrey Siegel of Biophotonics Center at Albert Einstein College of Medicine.Today we will show you a procedure for intravital photo switching and imaging of DRA two labeled cancer cells through the memory imaging window. We use this procedure in our laboratory to study influence of the tumor microenvironment on the motility of tumor cells. So let's do it Wearing latex gloves.Begin the fabrication of the mammary imaging window, or MIW by heating up a five to 10 centimeter tissue culture dish and creating a curved surface by pushing a rounded hard object such as a one inch Dremel bit against the dish. Cut out the center of the dish using a heated razor blade. Now use a small cone-shaped dremmel bit to make a six to seven millimeter diameter hole in the center of the dome shaped plastic base.Make the edges of the plastic base completely smooth by sanding it with the Dremel and further filing it. File the top of the plastic base to make a flat surface for the glass cover slip. The diameter of the filed flat surface should be nine to 10 millimeters.Next, glue the eight millimeter circular glass cover. Slip onto the flattened surface using super glue and let it dry for 15 minutes. Using a heated 26 G needle, make eight suturing holes by puncturing from the outside to the inside of the base, the holes should be evenly distributed around the cover.Slip nwt 0.5 to one millimeter away from the edge of the cover slip. Wash the MIW with deionized water, and then again with 70%ethanol. Use a Q-tip to clean the glass until it is completely transparent.Use acetone to remove any remaining foggy spots from the glue vapor. Finally sterilize the MIW by exposing it to ultra filet light for three hours on each side. Before inserting the MIW, the area around the fourth nipple should have developed a small five to seven millimeter diameter tumor as a result of the injection of tumor cells.In part one Check to make sure that the tumor is not visibly necrotic. There should be intact skin with hair on the area around it. To begin, prepare a sterile space for the surgery by laying down a piece of sterile cloth inside a sterile hood.Put a piece of sterile gauze in the middle and gather the items you will need for the surgery. You'll need a pair of small and a pair of large dissection, spring scissors, micro dissecting tweezers and forceps, and a needle holder After anesthetizing the mouse with 2.5%averting, use a small animal shaver to shave the area above the tumor. Use their hair removal cream to remove the rest of the hair and clean the skin.Using ethanol dipped Q-tips. Transfer the animal onto the sterile gauze and apply ophthalmic ointment to the eyes to keep them from drying out and becoming infected. Sterilize the skin with Betadine and then clean it with 70%ethanol.With the annual and sterile surgical cloth, pull the skin immediately medial to the nipple using forceps and cut an approximately two millimeter incision. Separate the underlying mammary fat pad from the skin using dissection, scissors, and forceps. When separated, the skin and hole stretch to a size that will accommodate insertion of the MIW.Insert the MIW such that there is skin on top of the MIW base and suture it in place. Use tissue adhesive or cyanoacrylate to fill in the suturing holes and secure the MIW to the skin. The animal should now be left to recover for the next three to four days before the first imaging session takes place.In preparation for the imaging session, we can describe the imaging box to make use of the imaging box. First, anesthetize the animal with isof fluorine and apply ophthalmic ointment to the eyes. Attach the isof fluorine exhaust from the anesthesia machine to the front of the imaging box.On the outside of the inlet hole. Attach a second tube to the outlet hole of the imaging box. This tube should be attached to the gas filter and then to the vacuum.Next, place the animal down so that the MIW is facing the bottom. Hold the box and adjust the bottom doors so that the MIW base is held and immobilized between them. The MIW base should be at the same level as the imaging box doors.While the MIW cover slip should be just a few millimeters below this level, make sure that the positioning of the MIW between the bottom sliding doors ensures that the cover slip lies flat parallel to the bottom doors of the box. This will allow for proper focusing lower the microscope objective to allow space to put the box on top of the microscope stage. Adjust the stage so that the MIW cover slip is above the objective.We can now focus the objective and start imaging. The imaging procedure described here is for the Leica SP five confocal microscope and should be used as a guide to optimize the experiment with a particular microscope being used. Begin by observing the tumor through the eyepiece with a green fluorescent filter.Locate major blood vessels that are visibly flowing and laying flat in the same focal plane. Position one of the vessels in the center of the field and switch to PMT detection. We will set up sequential imaging collection for two channels.One channel uses the 488 nanometer laser line and collects scattering from the extracellular matrix and emission from the green form of dendra two. The second channel uses the 543 nanometer laser line and collects emission from the red form of dendra.Two. Collect 3D images before photo switching.Next, use the region of interest scan option to photo switch a chosen population of cells with the 405 nanometer laser line. Collect emission from the red form of the protein in order to monitor the switching. Now collect post switching 3D images by the same method that pre switching images were collected.Alright, so with the use of 20%laser power in the UV to go or convert this area to photo switch more than one region in the tumor, take pictures through the oculars using a digital camera in both the green and red channels. This will help with orientation during subsequent imaging sessions. In this image, one observes a rectangular photo switch, region oriented orthogonally relative to the blood vessel, which exhibits no fluorescence.Non-photo switch cells appear green while the scattering from the extracellular matrix is purple. The image is a maximum intensity projection of four images across the Z axis. We've just shown you how to photo switch and image RA tool labeled cells through the memory imaging window.Using this procedure is important to compare behaviors of tumor cells in different areas of tumor over days. So that's it. Thanks for watching and good luck with your experiment.

dendra2-photoswitching-through-the-mammary-imaging-window

Research

JoVE Journal

Biology

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Cranial Window-based Intravital Imaging in a Mouse Model: An Imaging Technique to Study the Behavior of Pre-injected Cancer Cells in the Brain of a Murine Model

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Longitudinal Intravital Microscopy Using a Mammary Imaging Window with Replaceable Lid

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The present protocol describes a facile technique for the intravital imaging of the lactating mouse mammary gland by laser scanning confocal and multiphoton microscopy.

Intravital Subcellular Microscopy of the Mammary Gland

We present a protocol for the surgical implantation of a stabilized indwelling optical window for subcellular-resolution imaging of the murine pancreas, allowing serial and longitudinal studies of the healthy and diseased pancreas.

Stabilized Window for Intravital Imaging of the Murine Pancreas

Cortical blood flow dynamics can be studied in vivo by imaging fluorescent dextran dyes injected into the tail vein of rodents with 2-photon microscopy. This video shows how to image blood flow dynamics in neocortex of mice through a glass-covered cranial window preparation.

A Method for 2-Photon Imaging of Blood Flow in the Neocortex through a Cranial Window

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications, and how it should be maintained as required to obtain maximum image-forming capacity and resolution. The components of the microscope are described in detail here.

Major Components of the Light Microscope

Mammary tumor cells expressing luciferase are implanted subcutaneously in mice and visualized using optical imaging to monitor tumor growth and development non-invasively in a longitudinal study.

In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

Learn intravital photoswitching of Dendra2-labeled tumor cells in mouse mammary fat pad using a specialized imaging window. This protocol enables tracking metastatic behavior over several days.

Dendra2 Photoswitching through the Mammary Imaging Window

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